The ability to diagnose bacterial infection is an important aspect of modem medicine. Frequently, a patient's symptoms alone are insufficient to indicate either that an infection is the cause of an illness or to identify the specific pathogen when bacterial infection is suspected. Diagnostic methods are constantly being devised to more accurately evaluate bacterial infections.
Many techniques for developing methods for diagnosing bacterial infection involve the approach of analyzing a specimen taken from a patient for a telltale characteristic. For example, the specimen can be analyzed for the pathogen directly, or for a biochemical product that indirectly indicates the presence of the pathogen in the patient's body. Biopsy or similarly difficult or painful sampling procedure is sometimes required to obtain the required specimen. This is often necessary for digestive system infections which target organs located deep within the body.
Recent developments in the field of biotechnology permit diagnosis for infection by analyzing non-invasively obtained specimens, such as blood serum, urine, mucous discharges and the like. A favored traditional approach to developing such diagnostic methods typically entails several steps that include (i) biochemically mapping the infecting organism in detail; (ii) isolating and/or synthesizing specific biochemical fragments identified by the mapping or antigens produced by the organism; (iii) exposing a specimen from the patient to the specific biochemical fragments or antigens; (iv) testing for a reaction to the exposure; and (v) confirming that the specific biochemical fragments or antigens are exclusive to the infecting organism. Step (i) usually requires an exhaustive and thorough analysis of the bacteria and how it affects the patient. Step (v) is necessary to rule out false positive diagnoses based upon response to extraneously introduced fragments. Generally these steps (i) and (v) are complicated, require a high level of biomedical skill, and can take a very long time to accomplish.
The traditional technique of developing bacterial infection diagnostic procedures is further hindered by the inherent nature of antibodies to remain permanently in the body. Diagnostic procedures developed in the customary way often determine interaction between specific antibodies and antigens previously found to be associated with the infecting organism. Because antibodies can remain detectable in the host indefinitely, a convalescent patient who was previously successfully treated for the infection but is not presently viably infected is likely to generate an interactive response when tested by such a conventionally developed diagnostic procedure. The procedure cannot distinguish between the existence of a viable infection and a not presently symptomatic (i.e., "inactive") infection. Thus, the traditional approach suffers from the drawback that it ultimately might not lead to a successful result.
A simple and efficient method for developing highly discriminatory and sensitive diagnostic procedures for bacterial infection is very desirable. Such a method which includes the ability to easily develop diagnostic procedures that can distinguish active infections from inactive infections as well as from non-infections has been discovered. The new method has been demonstrated by example in the context of developing a procedure for diagnosing active infections of Helicobacter pylori.
Helicobacter pylori (hereinafter, "Hp") is a rod-shaped bacterium which infects the gastric mucosa. Infection by Hp is a leading cause of certain gastric diseases such as duodenal ulcers and gastric ulcers. The bacterium also has been implicated in development of gastric cancer. Symptoms of Hp infection vary from mild to severe and can include abdominal pain, nausea, indigestion, gas and bloating. Some of these symptoms are similar to those of unrelated health conditions such as simple indigestion and chronic dyspepsia. Thus a method for detecting Hp should be both sensitive and discriminatory to accurately identify the nature of a patient's illness.
Various methods of diagnosing Hp infection have been developed. For example, Hp can be detected by culture assay or by the Campylobacter-like organism ("CLO") test described in U.S. Pat. No. 4,748,113, incorporated herein by reference. Both test types require obtaining a sample by highly invasive, endoscopic biopsy. Culturing methods additionally are time consuming and produce variably successful diagnoses. Faster CLO analysis detects urease produced by Hp. However, the enzyme can be present due to causes other than Hp. Thus CLO testing usually is not adequately specific for detecting the presence of the Hp pathogen. Certain non-invasive Hp diagnostic methods have also been developed. These include breath tests that determine the presence of ureas associated with Hp infection. Such methods detect radioactive and non-radioactive carbon isotope-bearing urea compounds. Difficulty introduced by the need to control hazardous materials is an obvious shortcoming of the radioactive carbon test. Breath tests generally are reported to be adequately sensitive and reliably specific for Hp. Unfortunately, conduct of the such tests is labor intensive and requires many minutes of patient participation. These characteristics render breath tests impractical for screening the very large potential patient populations which are susceptible to Hp infection. Also, specialized equipment not usually installed in most present-day clinical laboratories is necessary to carry out breath tests.
Enzyme-linked immunosorbent assay ("ELISA") is a serologic test applicable to Hp detection. ELISA analysis would seem well suited to Hp diagnosis because it is fast, non-labor intensive, uses samples obtained quickly without discomfort or inconvenience of the patient, and employs inexpensive equipment and materials. Unfortunately, ELISA-based tests are not as sensitive as other diagnostic methods. Furthermore, ELISA tests analyze for antibodies produced by Hp infection rather than the presence of the bacteria directly. The antibodies can remain present in the patient long after Hp has been eliminated, for example by prior antibiotic treatment. Consequently, ELISA analysis cannot reliably distinguish between a viable Hp infection and a previously treated, presently non-symptomatic, inactive infection.
There is great need for a very sensitive and highly specific diagnostic procedures capable of detecting bacterial infections generally, and Hp infection in particular. The present invention now provides a method that can be applied to diverse pathogen-patient systems to develop new diagnostic procedures for detecting bacterial infections. The new diagnostic procedure development method can be applied in situations involving an infecting bacterial pathogen which engenders an immune response in an infected patient animal. Accordingly, there is provided a method of developing a diagnostic procedure for detecting infection of an animal by an infecting bacterial pathogen comprising the steps of:
(A) verifying that the infecting pathogen is capable of generating an immune response to a specimen bearing antibodies from an animal known to have a viable infection of the infecting bacterial pathogen; PA1 (B) Providing a plurality of groups of at least one protein encoded by a gene of a random fragment of the genomic DNA of the infecting bacterial pathogen; PA1 (C) obtaining a known specimen bearing antibodies from an animal known to have a viable infection of the infecting bacterial pathogen; PA1 (D) separately contacting the groups of proteins with a portion of the known specimen; PA1 (E) identifying as a positively responsive group, each group of proteins which evokes a positive immune response to contact with the known specimen; PA1 (F) obtaining a first control specimen bearing antibodies from an animal known to be naive with respect to infection by the infecting bacterial pathogen; PA1 (G) separately contacting positively responsive groups of proteins with a portion of the first control specimen; and PA1 (H) identifying as a selectively responsive group, each of the positively responsive groups of proteins which evokes no immune response to contact with the first control specimen. PA1 breaking up the whole pathogen into random portions; PA1 obtaining a classed sample from a patient known have a viable infection of the infecting bacterial pathogen; PA1 testing by immunoassay each of the random portions for an immune response to the classed sample; and PA1 observing a positive immunoassay result for at least some of the portions. PA1 (A) verifying that the infecting pathogen is capable of generating an immune response to a specimen bearing antibodies from an animal known to have a viable infection of the infecting bacterial pathogen; PA1 (B) Providing a plurality of groups of at least one protein encoded by a gene of a random fragment of the genomic DNA of the infecting bacterial pathogen; PA1 (C) obtaining a known specimen bearing antibodies from an animal known to have a viable infection of the infecting bacterial pathogen; PA1 (D) separately contacting the groups of proteins with a portion of the known specimen; PA1 (E) identifying as a positively responsive group, each group of proteins which evokes a positive immune response to contact with the known specimen; PA1 (F) obtaining a first control specimen bearing antibodies from an animal known to be naive with respect to infection by the infecting bacterial pathogen; PA1 (G) separately contacting positively responsive groups of proteins with a portion of the first control specimen; PA1 (H) identifying as a selectively responsive group, each of the positively responsive groups of proteins which evokes no immune response to contact with the first control specimen; PA1 (I) obtaining a second control specimen bearing antibodies from an animal known to be convalescent with respect to infection by the infecting bacterial pathogen; PA1 (J) separately contacting selectively responsive groups of proteins with a portion of the second control specimen; and PA1 (K) identifying as a discriminatingly responsive group, each of the selectively responsive groups of proteins which evokes no immune response to contact with the second control specimen.
In a particular aspect of this invention, a method for verifying the immune response has been identified. The verifying steps include:
The present invention further provides a novel diagnostic procedure for detecting bacterial infection in a patient animal developed by the above method. The new diagnostic procedure features the ability to use minimally invasively obtained specimens from a patient to differentiate between a viable, convalescent infection and naive states with respect to infection by a pathogen of interest. Hence, the diagnostician can determine whether those cases in which the pathogen has been eliminated from the patient although long-lived antibodies remain resident. Additionally, the novel diagnostic procedure developed according to the present invention advantageously is easy to implement, uses commonly available laboratory materials, provides a fast result and therefore is amenable to screening large numbers of potential patients rapidly and inexpensively. Still further according to this invention there is provided a method of diagnosing a viable infection of an animal by an infecting bacterial pathogen comprising contacting blood serum from the animal with a selectively responsive protein and/or a discriminatingly responsive protein.
There is especially provided such a diagnostic method for the detection of Helicobacter pylon infection.
Through application of the novel diagnostic method development procedure there can be obtained a composition suitable for use in diagnosing active bacterial infections. Thus there are also provided compositions for use in a diagnostic procedure for detecting an infection of an animal by an infecting bacterial pathogen that engenders an immune response wherein a composition comprises a selectively responsive protein and another composition comprises a discriminatingly responsive protein. The selectively responsive protein and discriminatingly responsive protein are proteins encoded by fragments of the genomic DNA of the infecting bacterial pathogen. These compositions can be obtained by a method comprising the steps of:
Appreciating that a key ingredient of the composition useful for detecting bacterial infections is a protein encoded by the genomic DNA of the infecting pathogen, there has been discovered a way to produce colonies of mutant organisms with the ability to produce selectively responsive and discriminatingly responsive proteins. Thus there is also provided a method of producing cloned host bacteria capable of producing protein useful for diagnosing a patient animal for infection by an infecting bacterial pathogen that engenders an immune response. And yet further, there is provided a cloned host bacteria for making a reagent of a diagnostic procedure for detecting such infections. The cloned host bacteria of a species other than the infecting bacterial pathogen comprises a transfected fragment of genomic DNA of the infecting bacterial pathogen that encodes a selectively responsive protein or a discriminatingly responsive protein.